1、Designation: E3178 18Standard Practice forEvaluating Static and Cidal Chemical Decontaminantsagainst Bacillus Spores using Centrifugal Filtration Tubes1This standard is issued under the fixed designation E3178; the number immediately following the designation indicates the year oforiginal adoption o
2、r, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice is used to quantify the efficacy of liquid orsolid decontaminant
3、s on Bacillus spores dried on the surface ofcoupons made from porous and non-porous materials. Thispractice can distinguish between bactericidal and bacteriostaticchemicals within decontamination mixtures. This is importantbecause many decontaminants contain both reactive com-pounds and high concent
4、rations of bacteriostatic surfactants.All test samples are directly compared to pre-neutralizedcontrols, un-inoculated negative growth controls, and solutioncontrols on the same day as the test in order to increasepractical confidence in the inactivation data.1.2 This procedure should be performed o
5、nly by thosetrained in microbiological techniques, are familiar with anti-microbial (sporicidal) agents and the application instructions ofthe antimicrobial products.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This s
6、tandard may involve hazardous materials,operations, and equipment. This standard does not purport toaddress all of the safety concerns, if any, associated with itsuse. It is the responsibility of the user of this standard toestablish appropriate safety, health, and environmental prac-tices and deter
7、mine the applicability of regulatory limitationsprior to use.1.5 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendation
8、s issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 CFR Standards:3,4Title 7 (Agricu
9、lture) CFR, Part 331 Possession, Use, andTransfer of Select Agents and ToxinsTitle 9 (Animals and Animal Products) CFR, Part 121 Pos-session, Use, and Transfer of Select Agents and ToxinsTitle 42 (Public Health) CFR, Part 73 Select Agents andToxins3. Terminology3.1 For definitions of terms used in t
10、his Practice, seeTerminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 decontaminant, na physical or chemical agent orprocess that inactivates pathogenic or potentially pathogenicmicroorganisms in or on surfaces or objects.3.2.2 endospore, na dormant, robust and non-metabolicall
11、y active structure produced by certain types ofbacteria from the Firmicutes phylum.3.2.3 exosporium, nthe outermost structural layer of mac-robacillus spores including Bacillus anthracis, Bacillus th-uringiensis and Bacillus cereus.3.2.4 macrobacillus, na Bacillus species that producesendospores tha
12、t possess an exosporium including Bacillusanthracis, Bacillus thuringiensis and Bacillus cereus.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.C
13、urrent edition approved Oct. 1, 2018. Published January 2019. DOI: 10.1520/F3178182For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary pag
14、e onthe ASTM website.3United States Select Agent Program administered by the Center for DiseaseControl (CDC) and/or the United States Department ofAgriculture (USDA),Animaland Plant Health Inspection Service (APHIS)https:/www.selectagents.gov/4https:/www.access.gpo.govCopyright ASTM International, 1
15、00 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and
16、Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.2.5 microbacillus, na Bacillus species that producesendospores that do not possess an exosporium includingBacillus subtilis and Bacillus atrophaeus.4. Summary of Practice4.1 For the purpose of this
17、practice a decontaminant can beinterpreted to include liquids and solids that inactivate mi-crobes. This practice quantitatively evaluates the efficacy ofdecontaminants on coupons contaminated with Bacillus spores(pathogenic and non-pathogenic strains). Spores are dried oncoupon surfaces. The spore-
18、inoculated coupons are then trans-ferred to Amicon5Ultra-15 Centrifugal Filter Units withUltracel-100 membranes (filter units) contained in sterile50-mL conical tubes (1, 2).64.2 Coupon material is selected according to the claims orintended use of the decontaminant. Coupons may be made ofany materi
19、al - hard, flexible, porous, non-porous, metallic, ornon-metallic. Flat (2 cm 2 cm) coupons are preferred;however, non-flat coupons and smaller coupons can be testedusing this practice. Coupons that have been tested are 0.1-0.3cm thick. Check that the coupons are thin enough to fit withinthe filter
20、units mentioned in 4.1.4.3 Filter units contained in sterile 50-mL conical tubes (1,2) allows for greater flexibility in coupon material selectioncompared to microfuge tubes because these larger containerswill accommodate materials that are difficult to manufacture inextremely small sizes including
21、fabrics and painted surfaces.4.4 Filter units contained in sterile 50-mL conical tubes (1,2) allow the spore-inoculated coupons, which may contain bothcidal reactive compounds and static compounds includingsurfactants, to be submerged and then be filtered away after thetest. The membranes also allow
22、 for spores to be washed freefrom any residual chemicals that might have bound to the sporesurfaces. Effectively, all the inoculated spores, including thosebound to the coupons, can be recovered in the filter unit andassayed after filtering and washing the spore-inoculated cou-pons. Such data provid
23、e confidence that spores have beeninactivated and the results are not merely the result of staticactivity.4.5 Contaminated test coupons are subjected to decontami-nation procedures. Control coupons are treated in an identicalmanner to test coupons but with a pre-neutralized decontami-nant. Recovery
24、of viable spores from pre-neutralized decon-taminant on the same day(s) as the test samples providesgreater confidence in the test data by eliminating time as a testvariable.4.6 Spores suspended in an aqueous medium represent thespore recovery reference for calculating spore survival afterdecontamin
25、ation treatment and analysis.4.7 Spore extraction percentage is calculated by dividing thenumber of spores recovered from each spore-inoculated controlcoupon by the number of spores recovered from the aqueousmedium controls.4.8 The number of culturable surviving spores from decon-tamination tests is
26、 divided by the extraction percentage todetermine the number of surviving spores in CFU mL-1andaccount for any surviving spores not removed from the couponduring extraction. This spore concentration is then multipliedby 10 mL to give the total number of spores surviving (CFU)from each test sample. A
27、 log10transformation of the totalsurviving spores is then performed (log10(total CFU + 1).5. Significance and Use5.1 The practice can be used to evaluate coupon materials ofany composition, insofar as the coupon can be small enough tofit inside filter units mentioned in 4.1.5.2 This practice defines
28、 procedures that are quantitative,scalable, rapid, sensitive, and safe, while minimizing labor andaddressing statistical confidence (1, 2).5.2.1 QuantitativeThe total number of spores per couponis determined by dilution-plating, and all spores remaining onthe coupon are assayed for activity in the e
29、xtraction tube toprovide confidence that all the spores were accounted for.5.2.2 Statistical ConfidenceThe use of five independentpreparations of spore inocula for a statistical n of 5.5.2.3 SensitivityAllows for complete detection of all cul-turable spores inoculated on a coupon, including the spor
30、es thatremain attached to the coupon.5.2.3.1 The limit of detection is dependent on the cultur-ability of fully matured spores to germinate, outgrow anddivide in the presence of the extraction medium (1% tryptic soybroth, 100 mM L-Alanine, 1 mM inosine, 0.05% Tween 80)and/or on tryptic soy agar.5.2.
31、3.2 Results presented in Refs (1, 3) (and currentlyunpublished results) indicate that these media, combined withthe test temperatures and conditions described herein willgenerate results with a high level of practical confidence fordetecting culturable Bacillus spores.5.2.4 SafetyInoculated coupons
32、are contained within filterunits.5.2.5 Simplicity of TestingTests and extractions are per-formed in the same filter unit to minimize coupon handlingsteps.5.2.6 Scalable and RapidA maximum of 36 samples canbe processed in1hbytwotechnicians; a total of 300 sampleshave been processed by six technicians
33、 in 5 h (1, 2).5.2.7 Wide application for numerous Bacillus species andstrains. The method has also been modified and used forvegetative bacteria and viruses as well (1, 2).6. Apparatus6.1 Autoclave.6.2 Shaking Incubator, capable of maintaining temperatureat 62 C within a minimum temperature range o
34、f 25-37 C.6.3 Gneral Purpose Microbiological Incubator (62 C).6.4 Phase-Contrast Microscope, oil immersion with magni-fication 100.6.5 Centrifuge, capable of 3,100xg that can hold aswinging-rotor bucket for 50-mL conical tubes.5Trademarked by Millipore, Billerica, MA, USA, UFC90100966The boldface nu
35、mbers in parentheses refer to the list of references at the end ofthis standard.E3178 1826.6 Water Bath, capable of maintaining temperature at62 C within a minimum temperature range of 50-65 C.6.7 Single-tube Vortex Mixer.6.8 Multi-tube Vortex Mixer.6.9 Analytical Balance.6.10 Ultra-Low Freezer, set
36、 at -60 C.6.11 Stopwatch or Electronic Timer.6.12 Manual or Electronic Pipettes.6.13 Bio-Safety Cabinet (BSC).6.14 Appropriate PPE, for example, gloves, safety glasses,lab coats, etc. (4).6.15 Pipettes, 200 L and 1 mL.7. Reagents and Materials77.1 Reagents:7.1.1 Bacillus anthracis, cereus and thurin
37、giensisacquisition, holding, preparations and/or testing of virulentstrains require CDC orAPHIS registration in the United States.Strains can be obtained fromATCC, the Bacillus Genetic StockCenter, BEI resources repository at National Institute ofHealth, or the Unified Culture Collection (UCC) at US
38、AMedical Research Institute of Infectious Disease.8,97.1.1.1 B. anthracis strain examples include virulent Ames(UCC BACI387), attenuated Sterne (UCC BACI397), andattenuated Sterne (UCC BACI056).7.1.1.2 B. cereus strain examples include a type strain(ATCC 10792), E33L (UCC BACI267), and 03BB102 (UCCB
39、ACI234).7.1.1.3 B. thuringiensis strain examples include Al Hakam(UCC BACI229), cry- HD-1 (Bacillus Genetic Stock CenterID4A12).7.1.2 Tryptic Soy Broth (TSB).7.1.3 Tryptic Soy Agar (TSA).7.1.4 Nutrient Broth (NB).7.1.5 Tween-80. 0.1%, 3% and 20% stock solutions ofTween-80 suspended in deionized wate
40、r.7.1.6 L-Alanine.7.1.7 Inosine.7.1.8 Sporulation Broth0.8% (w/v) Nutrient broth or2.5% (w/v) Nutrient broth and salts, as defined in Table 1,pH7 (see Appendix X1 for preparation instructions) (3).7.1.9 Extraction BufferpH 7, as defined in Table 2 (4, 5).7.1.10 pH-adjusted Bleach0.6% (v/v) hypochlor
41、ite,0.2% (v/v) glacial acetic acid, pH 6.5 and 7.0 within 1 hthat it is mixed. The pH will gradually drop to pH 4.0 and5.0 after storage for 7 d.7.1.11 90% (v/v) Ethanol.7.1.12 Sodium Thiosulfate (STS)7.2 Materials:7.2.1 Amicon Ultra-15 Centrifugal Filter Units withUltracel, 100K membranes (Millipor
42、e, Billerica, MA, USA,UFC9010096) (filter units).7.2.2 Sterile 50-mL Conical Tube.7.2.3 Baffled Flasks.7.2.4 Sterile Petri Dishes.7.2.5 50-mL Conical Tube and Microfuge Tube Racks.7.2.6 Pipette Tips.7.2.7 Parafilm10.7.2.8 L-shaped Sterile Spreaders.7.2.9 1.5-mL Sterile Microfuge Tubes.7.2.10 Coupon
43、MaterialsAll coupon materials must be astandardized surface area, preferably flat, 2 cm2cm;however, it is understood that not all materials are easilyadaptable to these size constraints.7.2.11 Sterile Forceps.7.2.12 Sodium Thiosulfate (STS).8. Hazards8.1 It is the responsibility of the individual us
44、er(s) of thispractice to follow all safety guidelines and to be knowledge-able about these procedures. Individual users should consulttheir safety authority and establish detailed safety plans andrisk assessments prior to using this practice. Users are stronglyurged to consult the Biosafety in Micro
45、biological and Biomedi-cal Laboratories (4).7Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Do
46、rset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.8Virulent strains require CDC (cross reference citation in 2.2) or APHIS (crossreference citation in 2.2) registration in the United States.9If you are aware of alternative
47、suppliers, please provide this information toASTM International Headquarters. Your comments will receive careful consider-ation at a meeting of the responsible technical committee,1which you may attend.10Trademarked by Bemis Company, Inc. Neenah, WI 54956TABLE 1 Sporulation Broth (pH 7)Reagent Final
48、 ConcentrationNutrient Broth 25 g L-1(2.5%) or 8 g L-1(0.8%)KH2PO4(Molecular Wt 136.04) 2.15 g L-1(15.8 mmol L-1)K2HPO4(Molecular Wt 174.18) 4.35 g L-1(25 mmol L-1)CaCl22H2O (Molecular Wt 147.01) 0.15 g L-1(1 mmol L-1)MnCl22H2O (Molecular Wt 197.9) 0.016 g L-1(0.1 mmol L-1)MgCl2(Molecular Wt 95.21)
49、0.095 g L-1(1 mmol L-1)ZnCl2(Molecular Wt 136.3) 0.068 g L-1(0.05 mmol L-1)FeCl36H2O (Molecular Wt 270.3) 0.0003 g L-1(0.001 mmol L-1)Sterile, 18 M-cm water Add water to 1 L final volumeTABLE 2 Extraction Buffer (pH 7)AReagent Final Concentration1X Extraction BufferTryptic Soy Broth 10 g L-1(1%)L-Alanine (Molecular Wt 89.09) 8.91 g L-1(100 mmol L-1)Inosine (Molecular Wt 268.23) 2.68 g L-1(1 mmol L-1)Tween 80 (Polysorbate 80) 0.5 mL L-1(.05%)Sterile, 18-megaohm water Add water to 1 L final volume2X Extraction BufferTryptic Soy Broth 20 g L
